Project Summary Our laboratory discovered in 1980 that methylation of DNA (epigenetic modification) affected gene expression and cell differentiation. High-throughput sequencing and the unexpected outcomes from The Cancer Genome Atlas and other projects show that many mutations in cancer are in genes that modify the epigenome. This has validated our long-term hypothesis that abnormal epigenetic processes are major contributors to human cancer and offer novel therapeutic opportunities. Understanding how the epigenome is changed in cancer requires an integrated approach, which we have developed over the last five years. We wish to determine the mechanisms by which key features such as DNA methylation, nucleosome positioning, and histone modifications influence each other. We then will determine how DNA methylation inhibitors (DNMTi?s) work. This grant is designed to use our powerful new NOMe-seq technology to understand the relationship between DNA methylation and nucleosomal positioning; to use knock-down and other approaches to examine the effects of altering both chromatin-remodeling and histone-modifying enzymes on the epigenome as a whole; and to understand how the epigenome controls endogenous retroviruses (ERVs). We will study how the epigenome is altered in human cancer, characterizing changes not only in gene promoter regions but also in enhancer and insulator regions and in genes themselves. Major questions to be addressed include 1) Why are there so many mutations in chromatin modifiers, and what are the effects of these mutations on the structure of the epigenome? 2) What are the functional consequences of activating the expression of cancer/testis genes by 5-azanucleoside? 3) What double-stranded RNAs are activated by 5-azanucleosides and how do these relate to cellular responses? 4) Can we design combinations of epigenetic drugs which might increase the effectiveness of 5-azanucleoside treatment? and 5) Can cryo-EM help to visualize complexes relevant to chromatin structure and functions? We will also study the roles of TET enzymes, which oxidize 5- methylcytosine to 5-hydroxymethylcytosine and require vitamin C as a cofactor, and the enzymes G9A and SETDB1, which methylate histone protein H3K9. Combinations that increase the expression of ERVs will be prioritized, because recent data strongly suggests that ERV expression may be linked to cellular changes, and quite possibly to clinical outcomes in cancer. Cancer patients are often deficient in vitamin C, implying that supplementation may markedly increase TET activity and patient response. Our approach is designed not only to understand the epigenome holistically but also to devise strategies which will increase patients' responses to drugs, perhaps by defining future rational drug combinations, in particular making use of DNMTi?s. Success of this project should have rapid mechanistic and translational impact, as DNMTi?s are FDA-approved or are currently in trials for cancer treatment.